A number of plant species have been targeted for use in “molecular farming” of mammalian proteins of pharmaceutical interest. These plant expression systems provide for low cost production of biologically active mammalian proteins and are readily amenable to rapid and economical scale-up (Ma et al. (2003) Nat. Rev. Genet. 4:794-805; Raskin et al. (2002) Trends Biotechnol. 20:522-531). Large numbers of mammalian and plant proteins require post-translational processing for proper folding, assembly, and function. Of these modifications, the differences in glycosylation patterns between plants and mammals offer a challenge to the feasibility of plant expression systems to produce high quality recombinant mammalian proteins for pharmaceutical use.
As peptides move through the endoplasmic reticulum (ER) and Golgi subcellular compartments, sugar residue chains, or glycans, are attached, ultimately leading to the formation of glycoproteins. The linkage between the sugar chains and the peptide occurs by formation of a chemical bond to only one of four protein amino acids: asparagine, serine, threonine, and hydroxlysine. Based on this linkage pattern, two basic types of sugar residue chains in glycoproteins have been recognized: the N-glycoside-linked sugar chain (also referred to as N-linked glycan or N-glycan), which binds to asparagine residues on the peptide; and the O-glycoside-linked sugar chain, which binds to serine, threonine, and hydroxylysine residues on the peptide.
The N-glycoside-linked sugar chains, or N-glycans, have various structures (see, for example, Takahashi, ed. (1989) Biochemical Experimentation Method 23—Method for Studying Glycoprotein Sugar Chain (Gakujutsu Shuppan Center), but share a common oligomannosidic core (see FIG. 29A). The initial steps in the glycosylation pathway leading to the formation of N-glycans are conserved in plants and animals. However, the final steps involved in complex N-glycan formation differ (Lerouge et al. (1998) Plant Mo. Biol. 38:31-48; Steinkellner and Strasser (2003) Ann. Plant Rev. 9:181-192). Plants produce glycoproteins with complex N-glycans having a core bearing two N-acetylglucosamine (GlcNAc) residues that is similar to that observed in mammals. However, in plant glycoproteins this core is substituted by a β1,2-linked xylose residue (core xylose), which residue does not occur in humans, Lewisa epitopes, and an α1,3-linked fucose (core α[1,3]-fucose) instead of an α1,6-linked core fucose as in mammals (see, for example, Lerouge et al. (1998) Plant Mol. Biol. 38:31-48 for a review) (see also FIG. 29B). Both the α(1,3)-fucose and β(1,2)-xylose residues reportedly are, at least partly, responsible for the immunogenicity of plant glycoproteins in mammals (see, for example, Ree et al. (2000) J. Biol. Chem. 15:11451-11458; Bardor et al. (2003) Glycobiol. 13:427-434; Garcia-Casado et al. (1996) Glycobiol. 6:471-477). Therefore removal of these potentially allergenic sugar residues from mammalian glycoproteins recombinantly produced in plants would overcome concerns about the use of these proteins as pharmaceuticals for treatment of humans.
A number of recombinantly produced glycoproteins currently serve as therapeutics or are under clinical investigation. Examples include the interferons (IFNs), erythropoietin (EPO), tissue plasminogen activator (tPA), antithrombin, granulocyte-macrophage colony stimulating factor (GM-CSF), and therapeutic monoclonal antibodies (mABs). The oligosaccharide component of the N-glycan structures of glycoproteins can influence their therapeutic efficacy, as well as their physical stability, resistance to protease attack, pharmacokinetics, interaction with the immune system, and specific biological activity. See, for example, Jenkins et al. (1996) Nature Biotechnol. 14:975-981.
Methods are needed to alter the glycosylation pattern in plant expression systems, specifically to inhibit plant-specific glycosylation of the eukaryotic core structure, to advantageously produce recombinant mammalian proteins with a humanized glycosylation pattern.